Umbilical Cord Artery Perivascular Stem Cell Injection Solution for Treating Ischemic Diseases and Preparation Method Thereof

Abstract

An umbilical cord artery perivascular stem cell injection solution for treating ischemic diseases including suspended umbilical cord artery perivascular stem cells and a solvent and a preparation method thereof are provided. The solvent may be umbilical cord blood-derived platelet-rich plasma or injectable PBS containing human serum albumin. The preparation method includes: cultivating an umbilical cord artery tissue to derive perivascular stem cells, performing an enzymatic digestion to obtain individual umbilical cord artery perivascular stem cells, followed by washing twice with PBS, and gently mixing evenly with the solvent to obtain the umbilical cord artery perivascular stem cell injection solution. The cells are taken from the umbilical cord of medical waste and have the characteristics of easy separation, fast proliferation and low immunogenicity.

What is claimed is: 
     
         1 . An umbilical cord artery perivascular stem cell injection solution for treating ischemic diseases, comprising:
 individual umbilical cord artery perivascular stem cells in a suspended state and a solvent.   
     
     
         2 . The umbilical cord artery perivascular stem cell injection solution for treating the ischemic diseases according to  claim 1 , wherein:
 the solvent is umbilical cord blood-derived platelet-rich plasma.   
     
     
         3 . The umbilical cord artery perivascular stem cell injection solution for treating the ischemic diseases according to  claim 2 , wherein:
 a content of the individual umbilical cord artery perivascular stem cells is 5×10 6 /mL.   
     
     
         4 . The umbilical cord artery perivascular stem cell injection solution for treating the ischemic diseases according to  claim 1 , wherein:
 the solvent is phosphate buffer saline (PBS) containing human serum albumin.   
     
     
         5 . The umbilical cord artery perivascular stem cell injection solution for treating the ischemic diseases according to  claim 4 , wherein:
 a content of the individual umbilical cord artery perivascular stem cells is 5×10 6 /mL; and   a content of the human serum albumin is 5 grams per 100 mL of the PBS.   
     
     
         6 . A method for preparing the umbilical cord artery perivascular stem cell injection solution for treating the ischemic diseases according to  claim 2 , comprising:
 cultivating an umbilical cord artery tissue to derive perivascular stem cells, then performing an enzymatic digestion on the perivascular stem cells to obtain the individual umbilical cord artery perivascular stem cells, followed by washing the individual umbilical cord artery perivascular stem cells twice with PBS, and gently mixing the individual umbilical cord artery perivascular stem cells with the umbilical cord blood-derived platelet-rich plasma evenly to obtain the umbilical cord artery perivascular stem cell injection solution.   
     
     
         7 . The method according to  claim 6 , wherein:
 a process for preparing the individual umbilical cord artery perivascular stem cells comprises:   collecting 7-15 cm of a fresh human umbilical cord after a delivery of a full-term newborn, and then transporting the fresh human umbilical cord to a laboratory with PBS containing 10% (w/v) penicillin-streptomycin and 1% (v/v) heparin at 4° C. on ice within 4 h;   taking out the fresh human umbilical cord in an ultra-clean workbench environment, squeezing blood out of the fresh human umbilical cord, and washing the fresh human umbilical cord with PBS repeatedly until the fresh human umbilical cord is free of blood and blood clots, then trimming two sections of the fresh human umbilical cord, cutting the fresh human umbilical cord along a long axis direction of a blood vessel in the fresh human umbilical cord, and separating an umbilical artery by a blunt dissection with forceps;   cutting the umbilical artery in a direction perpendicular to the umbilical artery to obtain tissue blocks of 1-2 mm 3  in size, spreading the tissue blocks evenly on a bottom of a 100 mm cell culture dish, and then placing the 100 mm cell culture dish in an incubator with 5% CO 2  and saturated humidity at 37° C. for 3 h;   then, slowly adding 5 mL of DMEM-LG medium to the 100 mm cell culture dish and gently placing the 100 mm cell culture dish in the incubator for a cultivation;   adding 3 mL of DMEM-LG medium to the 100 mm cell culture dish on the 3 rd  day of a first week of the cultivation and then adding 2 mL of DMEM-LG medium to the 100 mm cell culture dish on the 7 th  day of the first week of the cultivation; then, exchanging a half-volume of fluid in the 100 mm cell culture dish with fresh DMEM-LG medium twice in a second week of the cultivation, and then exchanging a whole-volume of the fluid twice in a third week of the cultivation, and during the third week of the cultivation, observing a spreading of adherent cells from an edge of the tissue blocks;   after 3 weeks of the cultivation, when the adherent cells grow to 80% confluence, removing the tissue blocks, then digesting the adherent cells with 0.05% (w/v) trypsin for a subculture; and   harvesting P3-P5 generation cells of the adherent cells for the umbilical cord artery perivascular stem cell injection solution.   
     
     
         8 . The method according to  claim 6 , wherein:
 a process for preparing the umbilical cord blood-derived platelet-rich plasma comprises:   adding 750 IU of heparin sodium to a 50 mL centrifuge tube to collect 40 mL of umbilical cord blood;   performing a centrifugation on the umbilical cord blood at a speed of 200 g for 10-15 min at room temperature to obtain a resultant liquid, then collecting an upper portion of the resultant liquid;   centrifuging the upper portion of the resultant liquid at a speed of 2000 g for 10 min to cause platelets in the upper portion of the resultant liquid to deposit at a bottom of the 50 mL centrifuge tube to obtain supernatant plasma; then, discarding part of the supernatant plasma, retaining a total 200 μL of the supernatant plasma and the platelets deposited at the bottom, and gently resuspending the total 200 μL of the supernatant plasma and the platelets to obtain the umbilical cord blood-derived platelet-rich plasma.   
     
     
         9 . The method according to  claim 6 , wherein:
 the individual umbilical cord artery perivascular stem cells is washed twice with the PBS without calcium and magnesium to obtain a cell precipitate, then the cell precipitate is resuspended with the umbilical cord blood-derived platelet-rich plasma, and then gently mixed evenly to obtain the umbilical cord artery perivascular stem cell injection solution; and   the umbilical cord artery perivascular stem cell injection solution is placed at 4° C. for a short-term storage, used within 12 h, and shaken gently before use.   
     
     
         10 . A method for preparing the umbilical cord artery perivascular stem cell injection solution for treating the ischemic diseases according to  claim 4 , comprising:
 cultivating an umbilical cord artery tissue to derive perivascular stem cells, then performing an enzymatic digestion on the perivascular stem cells to obtain individual umbilical cord artery perivascular stem cells, followed by washing the individual umbilical cord artery perivascular stem cells twice with PBS without calcium and magnesium to obtain a cell precipitate, and resuspending the cell precipitate with PBS containing 5% (w/v) human serum albumin to obtain a cell suspension solution, and then gently mixing the cell suspension solution evenly to obtain the umbilical cord artery perivascular stem cell injection solution;   wherein the umbilical cord artery perivascular stem cell injection solution is placed at 4° C. for a short-term storage, used within 12 h, and shaken gently before use.